Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft tumors led to an increased number of persistent DSBs. Oral administration of M3814 to two xenograft models of human cancer, using a clinically established 6-week fractionated radiation schedule, strongly potentiated the antitumor activity of IR and led to complete tumor regression at nontoxic doses. Our results strongly support DNA-PK inhibition as a novel approach for the combination radiotherapy of cancer. M3814 is currently under investigation in combination with radiotherapy in clinical trials.

Purpose: The purpose of this study was to determine strength of muscles involved with instrumentation (scaling) by dental hygienists and the additive effects of cellular (mobile) phone usage, as indicated by measurements of muscular force generation.Methods: A convenience sample of licensed dental hygienists currently in clinical practice (n=16) and an equal number of individuals not currently using devices/tools repetitively for work (n=16), agreed to participate in this pilot study. All participants completed a modified cell phone usage questionnaire to determine their use pattern and frequency. Upon completion of the questionnaire, participants' force production in six muscle groups was measured using a hand-held dynamometer. Descriptive statistics were used to analyze the data.Results: A total of 16 licensed dental hygienists (n=16) and 16 participants with no history of using tools/devices repetitively for work (n=16), comprised the experimental and control groups, repectively. The control group generated greater muscle force than the experimental group for the abductor pollicis longus (p=0.045). Significant differences were identified when comparing the low mobile phone users in the experimental group to the control group for the flexor pollicis brevis (p=0.031), abductor pollicis longus (p=0.031), and flexor digitorum (p=0.006), with the control group demonstrating higher muscle force. Years in clinical practice and mobile phone use was shown to have a significant effect on muscular force generation for the flexor pollicis brevis (F=3.645, df=3, p=0.020) and flexor digitorum (F=3.560, df=3, p=0.022); subjects who practiced dental hygiene the longest produced the least amount of muscle force.Conclusion: Results from this pilot study indicate there are no significant additive effects of cell phone use and dental hygiene practice on finger muscles used for instrumentation. However, results indicate that dental hygiene practice demonstrated significant effects on muscular strength as compared to individuals who do not use tools/devices repetitively for work. The small sample size may have impacted results and the study should be repeated with a larger sample.

ABSTRACT

The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as a virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as a cross-linker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of β1-integrins on the basolateral plasma membrane of epithelial cells and causes rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive β1-integrins and also of complete α3β1-integrin–laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and causes the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by cross-linking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild-type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results, we suggest that LecB has a role in disseminating bacteria along the cell-basement membrane interface.

IMPORTANCE Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes of nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interactions is not well understood. The significance of our research is in identifying cellular mechanisms of how LecB facilitates P. aeruginosa infection. We introduce LecB as a new member of the list of bacterial molecules that bind integrins and show that P. aeruginosa can move forward underneath attached epithelial cells by loosening cell-basement membrane attachment in a LecB-dependent manner.

ABSTRACT

Satellite viruses, most commonly found in plants, rely on helper viruses to complete their replication cycle. The only known example of a human satellite virus is the hepatitis D virus (HDV), and it is generally thought to require hepatitis B virus (HBV) to form infectious particles. Until 2018, HDV was the sole representative of the genus Deltavirus and was thought to have evolved in humans, the only known HDV host. The subsequent identification of HDV-like agents in birds, snakes, fish, amphibians, and invertebrates indicated that the evolutionary history of deltaviruses is likely much longer than previously hypothesized. Interestingly, none of the HDV-like agents were found in coinfection with an HBV-like agent, suggesting that these viruses use different helper virus(es). Here we show, using snake deltavirus (SDeV), that HBV and hepadnaviruses represent only one example of helper viruses for deltaviruses. We cloned the SDeV genome into a mammalian expression plasmid, and by transfection could initiate SDeV replication in cultured snake and mammalian cell lines. By superinfecting persistently SDeV-infected cells with reptarenaviruses and hartmaniviruses, or by transfecting their surface proteins, we could induce production of infectious SDeV particles. Our findings indicate that deltaviruses can likely use a multitude of helper viruses or even viral glycoproteins to form infectious particles. This suggests that persistent infections, such as those caused by arenaviruses and orthohantaviruses used in this study, and recurrent infections would be beneficial for the spread of deltaviruses. It seems plausible that further human or animal disease associations with deltavirus infections will be identified in the future.

IMPORTANCE Deltaviruses need a coinfecting enveloped virus to produce infectious particles necessary for transmission to a new host. Hepatitis D virus (HDV), the only known deltavirus until 2018, has been found only in humans, and its coinfection with hepatitis B virus (HBV) is linked with fulminant hepatitis. The recent discovery of deltaviruses without a coinfecting HBV-like agent in several different taxa suggested that deltaviruses could employ coinfection by other enveloped viruses to complete their life cycle. In this report, we show that snake deltavirus (SDeV) efficiently utilizes coinfecting reptarena- and hartmaniviruses to form infectious particles. Furthermore, we demonstrate that cells expressing the envelope proteins of arenaviruses and orthohantaviruses produce infectious SDeV particles. As the envelope proteins are responsible for binding and infecting new host cells, our findings indicate that deltaviruses are likely not restricted in their tissue tropism, implying that they could be linked to animal or human diseases other than hepatitis.

ABSTRACT

Dermonecrotic toxin (DNT) is one of the representative toxins produced by Bordetella pertussis, but its role in pertussis, B. pertussis infection, remains unknown. In this study, we identified the T-type voltage-gated Ca²⁺ channel Ca_V3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As Ca_V3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of B. pertussis. The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed.

IMPORTANCE Bordetella pertussis, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca²⁺ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of B. pertussis infection.

ABSTRACT

The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated adcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the adcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated adcAII strains. However, transformation of adcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated adcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of adcAII. Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype.

IMPORTANCE The Streptococcus pneumoniae capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of adcAII, a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the adcAII strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and adcAII, further investigation of which could further characterize mechanisms of capsule regulation.

ABSTRACT

The multifunctional nature of viral proteins is essentially driven by posttranslational modifications (PTMs) and is key for the successful outcome of infection. For influenza A viruses (IAVs), a composite pattern of PTMs regulates the activity of viral proteins. However, almost none are known that target the PB2 replication protein, except for inducing its degradation. We show here that PB2 undergoes a nonproteolytic ubiquitination during infection. We identified E3 ubiquitin ligases catalyzing this ubiquitination as two multicomponent RING-E3 ligases based on cullin 4 (CRL4s), which are both contributing to the levels of ubiquitinated forms of PB2 in infected cells. The CRL4 E3 ligase activity is required for the normal progression of the viral cycle and for maximal virion production, indicating that the CRL4s mediate a ubiquitin signaling that promotes infection. The CRL4s are recruiting PB2 through an unconventional bimodal interaction with both the DDB1 adaptor and DCAF substrate receptors. While able to bind to PB2 when engaged in the viral polymerase complex, the CRL4 factors do not alter transcription and replication of the viral segments during infection. CRL4 ligases catalyze different patterns of lysine ubiquitination on PB2. Recombinant viruses mutated in the targeted lysines showed attenuated viral production, suggesting that CRL4-mediated ubiquitination of PB2 contributes to IAV infection. We identified K29-linked ubiquitin chains as main components of the nonproteolytic PB2 ubiquitination mediated by the CRL4s, providing the first example of the role of this atypical ubiquitin linkage in the regulation of a viral infection.

IMPORTANCE Successful infection by influenza A virus, a pathogen of major public health importance, involves fine regulation of the multiple functions of the viral proteins, which often relies on post-translational modifications (PTMs). The PB2 protein of influenza A viruses is essential for viral replication and a key determinant of host range. While PTMs of PB2 inducing its degradation have been identified, here we show that PB2 undergoes a regulating PTM signaling detected during infection, based on an atypical K29-linked ubiquitination and mediated by two multicomponent E3 ubiquitin ligases. Recombinant viruses impaired for CRL4-mediated ubiquitination are attenuated, indicating that ubiquitination of PB2 is necessary for an optimal influenza A virus infection. The CRL4 E3 ligases are required for normal viral cycle progression and for maximal virion production. Consequently, they represent potential candidate host factors for antiviral targets.

ABSTRACT

The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C–DNase X complex mediates the ROS blockade was unknown. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia-induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia-mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation.

IMPORTANCE Ehrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies.

Drivers with blood-alcohol levels below legal limits cause 15% of all crash deaths that involve alcohol, a study in the March issue of the American Journal of Preventive Medicine finds.

To the Editor—A 30-day injectable form of buprenorphine branded as SublocadeTM (Buprenorphine XR SQ) was approved by the FDA in 2017. This medication is administered by a health care professional subcutaneously in the abdomen to treat opioid use disorder. This long-acting delivery system holds great promise for many patients who have barriers to taking daily transmucosal buprenorphine-containing medications such as those with poor adherence to a daily medication. It is beneficial for those who have difficulty safely storing their medications, including patients who have children in the home, unstable housing, or live with others who have a use disorder. This product is also an option for patients who prefer mono-product buprenorphine. As Buprenorphine XR SQ is administered directly by a health care professional, it does not contain the abuse-deterrent naloxone that some patients feel causes side effects.

There are two ways to acquire Buprenorphine XR SQ: 1) order product from the distributor (buy and bill); or 2) dispensed from a specialty pharmacy for a specific patient (specialty pharmacy) [1]. For the buy and bill option, the health care setting must be certified through the Risk Evaluation and Mitigation Strategy (REMS) program and adhere to dispensing regulations [2]. We found this challenging to implement in the outpatient setting, thus we pursued the specialty pharmacy option. It ultimately took us nearly one year to complete the process.

The following are the barriers we faced with our first attempt. As a controlled substance, the medication must be stored in a refrigerated lockbox. Before...

BACKGROUND Pregnant patients from rural counties of Western North Carolina face additional barriers when accessing comprehensive perinatal substance use disorders care at Project CARA as compared to patients local to the program in Buncombe County. We hypothesized regional patients would be less engaged in care.

METHOD Using a retrospective cohort design, univariate analyses (², t-test; P < .05) compared patients' characteristics, engagement in care, and delivery outcomes. Engagement in care, the primary outcome, was operationalized as: attendance at expected, program-specific prenatal and postpartum visits, utilization of in-house counseling, community-based and/or inpatient substance use disorders treatment, and maternal urine drug screen at delivery negative for illicit substances.

RESULTS Regional patients (n = 324) were more likely than Buncombe County patients (n = 284) to have opioid [209 (64.5%) versus 162 (57.0%)] or amphetamine/methamphetamine use disorders (25 [7.7%] versus 13 [4.6%]), but less likely to have cannabis use (19 [5.9%] versus 38 [13.4%]; P = .009) and concurrent psychiatric disorders (214 [66.0%] versus 220 [77.5%]; P = .002). Engagement at postpartum visits was the significantly different outcome between patients (110/221 [49.8%] versus 146/226 [64.6%]; P = .002).

LIMITATIONS Outcomes were available for 66.8% of regional and 79.6% of Buncombe County patients of one program in one predominately white, non-Hispanic region of the state.

CONCLUSION Contrary to our hypothesis, regional and Buncombe County women engaged in prenatal care equally. However, a more formal transition into the postpartum period is needed, especially for regional women. A "hub-and-spokes" model that extends delivery of perinatal substance use disorders care into rural communities may be more effective for engagement retention.

In plants, water use efficiency (WUE) is a complex trait arising from numerous physiological and developmental characteristics. Here, we investigated the involvement of circadian regulation in long-term WUE in Arabidopsis (Arabidopsis thaliana) under light and dark conditions. Circadian rhythms are generated by the circadian oscillator, which provides a cellular measure of the time of day. In plants, the circadian oscillator contributes to the regulation of many aspects of physiology, including stomatal opening, rate of photosynthesis, carbohydrate metabolism, and developmental processes such as the initiation of flowering. We investigated the impact of the misregulation of numerous genes encoding various components of the circadian oscillator on whole plant, long-term WUE. From this analysis, we identified a role for the circadian oscillator in WUE. It appears that the circadian clock contributes to the control of transpiration and biomass accumulation. We also established that the circadian oscillator within guard cells can contribute to long-term WUE. Our experiments indicate that knowledge of circadian regulation will be important for developing crops with improved WUE.

The aim of this study was to identify factors associated with the death of patients with COVID-19 pneumonia caused by the novel coronavirus SARS-CoV-2.

All clinical and laboratory parameters were collected prospectively from a cohort of patients with COVID-19 pneumonia who were hospitalised to Wuhan Pulmonary Hospital (Wuhan City, Hubei Province, China) between 25 December 2019 and 7 February 2020. Univariate and multivariate logistic regression was performed to investigate the relationship between each variable and the risk of death of COVID-19 pneumonia patients.

In total, 179 patients with COVID-19 pneumonia (97 male and 82 female) were included in the present prospective study, of whom 21 died. Univariate and multivariate logistic regression analysis revealed that age ≥65 years (OR 3.765, 95% CI 1.146-17.394; p=0.023), pre-existing concurrent cardiovascular or cerebrovascular diseases (OR 2.464, 95% CI 0.755-8.044; p=0.007), CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 (OR 3.982, 95% CI 1.132-14.006; p<0.001) and cardiac troponin I ≥0.05 ng·mL^–1 (OR 4.077, 95% CI 1.166-14.253; p<0.001) were associated with an increase in risk of mortality from COVID-19 pneumonia. In a sex-, age- and comorbid illness-matched case–control study, CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 and cardiac troponin I ≥0.05 ng·mL^–1 remained as predictors for high mortality from COVID-19 pneumonia.

We identified four risk factors: age ≥65 years, pre-existing concurrent cardiovascular or cerebrovascular diseases, CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 and cardiac troponin I ≥0.05 ng·mL^–1. The latter two factors, especially, were predictors for mortality of COVID-19 pneumonia patients.

Early recognition of longus colli calcific tendinitis can prevent unnecessary interventions including antibiotics and surgical procedures.

Electronic cigarette or vaping product use–associated lung injury (EVALI) is a newly emerging diagnosis in the United States, yet the incidence has surged greatly in the past year. With the trend of using electronic cigarettes (e-cigarettes) and vaping rising at an alarming rate among teenagers, many are resorting to friends, illicit drug dealers, and other informal sources to obtain their e-cigarettes, which is greatly contributing to the national outbreak of EVALI. The incidence of adolescents presenting with the constellation of respiratory, gastrointestinal, and constitutional symptoms characteristic of EVALI has been widely reported within the nation. We present one such case of an adolescent boy with a 2-year history of daily vaping who presented with nausea, vomiting, weight loss, and fever but lacked the respiratory symptoms that have been reported in the majority of EVALI cases reported thus far. Computed tomography scan of the abdomen and pelvis revealed an incidental finding of lung pathology characteristic of EVALI, prompting further workup and diagnosis of EVALI. In this case, it is demonstrated that the presentation of EVALI can be variable and is still poorly defined. The rising morbidity and mortality from EVALI reveal the importance of considering EVALI in all patients with a history of vaping or e-cigarette use, regardless of the presence or absence of respiratory symptoms.

OBJECTIVES:

Standing orders are an effective way to increase vaccination rates, yet little is known about how pediatricians use this strategy for childhood immunizations. We assessed current use of, barriers to using, and factors associated with use of standing orders for vaccination among pediatricians.

Causal analysis is a core function of safety programs. Although established protocols exist for conducting root cause analysis for serious safety events, there is limited guidance for apparent cause analysis (ACA) in health care. At our institution, through a novel facilitated ACA approach, we aim to improve safety culture and provide a clear approach to address precursor safety events and near-miss safety events. We define facilitated ACA as limited investigation (scope and duration) of a safety event that resulted in little to no harm. These investigations require fewer resources and focus on preventive strategies. Our facilitated ACA model, with an operational algorithm and structured process, was developed and implemented at our tertiary-care, freestanding, urban pediatric hospital in 2018. Sixty-four ACAs were completed, and 83% were identified with the algorithm. Process measures, including time from event reporting to ACA launch (median 3 days; interquartile range 2–6 days), are tracked. Patient safety consultants averaged 5 hours to complete a facilitated ACA. A median of 3 disciplines or departments participated in each facilitated ACA. Through an iterative process, we implemented a structured process for facilitated ACA, and the model’s strength includes (1) right event, (2) right team, (3) right analysis, and (4) right action plans. This novel facilitated ACA model may support organizational cause analysis and improve safety culture with higher-reliability processes.

The vertebrate limb serves as an experimental paradigm to study mechanisms that regulate development of the stereotypical skeletal elements. In this study, we simultaneously inactivated Sall4 using Hoxb6Cre and Plzf in mouse embryos, and found that their combined function regulates development of the proximal-anterior skeletal elements in hindlimbs. The Sall4; Plzf double knockout exhibits severe defects in the femur, tibia, and anterior digits, distinct defects compared to other allelic series of Sall4; Plzf. We found that Sall4 regulates Plzf expression prior to hindlimb outgrowth. Further expression analysis indicated that Hox10 genes and GLI3 are severely downregulated in the Sall4; Plzf double knockout hindlimb bud. In contrast, PLZF expression is reduced but detectable in Sall4; Gli3 double knockout limb buds, and SALL4 is expressed in the Plzf; Gli3 double knockout limb buds. These results indicate that Plzf, Gli3, and Hox10 genes downstream of Sall4, regulate femur and tibia development. In the autopod, we show that Sall4 negatively regulates Hedgehog signaling, which allows for development of the most anterior digit. Collectively, our study illustrates genetic systems that regulate development of the proximal-anterior skeletal elements in hindlimbs.

Repeated alcohol experiences can produce long-lasting memories for sensory cues associated with intoxication. These memories can problematically trigger relapse in individuals recovering from alcohol use disorder (AUD). The molecular mechanisms by which ethanol changes memories to become long-lasting and inflexible remain unclear. New methods to analyze gene expression within precise neuronal cell types can provide further insight toward AUD prevention and treatment. Here, we used genetic tools in Drosophila melanogaster to investigate the lasting consequences of ethanol on transcription in memory-encoding neurons. Drosophila rely on mushroom body (MB) neurons to make associative memories, including memories of ethanol-associated sensory cues. Differential expression analyses revealed that distinct transcripts, but not genes, in the MB were associated with experiencing ethanol alone compared to forming a memory of an odor cue associated with ethanol. Adult MB-specific knockdown of spliceosome-associated proteins demonstrated the necessity of RNA-processing in ethanol memory formation. These findings highlight the dynamic, context-specific regulation of transcription in cue-encoding neurons, and the lasting effect of ethanol on transcript usage during memory formation.

BEAF (Boundary Element-Associated Factor) was originally identified as a Drosophila melanogaster chromatin domain insulator-binding protein, suggesting a role in gene regulation through chromatin organization and dynamics. Genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes, suggesting a role in promoter function. This would be a nontraditional role for an insulator-binding protein. To gain insight into molecular mechanisms of BEAF function, we identified interacting proteins using yeast two-hybrid assays. Here, we focus on the transcription factor Serendipity (Sry-). Interactions were confirmed in pull-down experiments using bacterially expressed proteins, by bimolecular fluorescence complementation, and in a genetic assay in transgenic flies. Sry- interacted with promoter-proximal BEAF both when bound to DNA adjacent to BEAF or > 2-kb upstream to activate a reporter gene in transient transfection experiments. The interaction between BEAF and Sry- was detected using both a minimal developmental promoter (y) and a housekeeping promoter (RpS12), while BEAF alone strongly activated the housekeeping promoter. These two functions for BEAF implicate it in playing a direct role in gene regulation at hundreds of BEAF-associated promoters.

The way genes contribute to behavior is complicated. Although there are some single genes with large contributions, most behavioral differences are due to small effects from many interacting genes. This makes it hard to identify the genes that cause behavioral differences. Mutagenesis screens in model organisms, selective breeding experiments in animals, comparisons between related populations with different behaviors, and genome-wide association studies in humans are promising and complementary approaches to understanding the heritable aspects of complex behaviors. To connect genes to behaviors requires measuring behavioral differences, locating correlated genetic changes, determining when, where, and how these candidate genes act, and designing causative confirmatory experiments. This area of research has implications from basic discovery science to human mental health.

Key Points

Epstein-Barr virus (EBV) causes B cell lymphomas and transforms B cells in vitro. The EBV protein EBNA3A collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro. An EBNA3A deletion mutant EBV strain was recently reported to establish latency in humanized mice but not cause tumors. Here, we compare the phenotypes of an EBNA3A mutant EBV (3A) and wild-type (WT) EBV in a cord blood-humanized (CBH) mouse model. The hypomorphic 3A mutant, in which a stop codon is inserted downstream from the first ATG and the open reading frame is disrupted by a 1-bp insertion, expresses very small amounts of EBNA3A using an alternative ATG at residue 15. 3A caused B cell lymphomas at rates similar to their induction by WT EBV but with delayed onset. 3A and WT tumors expressed equivalent levels of EBNA2 and p16, but 3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, 3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. Transcriptome sequencing (RNA-seq) analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus 3A-infected tumors and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that the expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together, these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice.

IMPORTANCE The EBV protein EBNA3A is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro. In this study, we used a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A hypomorph mutant virus (3A) and wild-type EBV. The 3A virus caused lymphomas with delayed onset compared to the onset of those caused by WT EBV, although the tumors occurred at a similar rate. The WT EBV and EBNA3A mutant tumors expressed similar levels of the EBV protein EBNA2 and cellular protein p16, but in some cases, 3A tumors had less LMP1. Our analysis suggested that 3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK cell responses to EBV-infected tumors.

Simian-human immunodeficiency virus (SHIV) infection of rhesus monkeys is an important preclinical model for human immunodeficiency virus type 1 (HIV-1) vaccines, therapeutics, and cure strategies. SHIVs have been optimized by incorporating HIV-1 Env residue 375 mutations that mimic the bulky or hydrophobic residues typically found in simian immunodeficiency virus (SIV) Env to improve rhesus CD4 binding. We applied this strategy to three SHIV challenge stocks (SHIV-SF162p3, SHIV-AE16, and SHIV-325c) and observed three distinct outcomes. We constructed six Env375 variants (M, H, W, Y, F, and S) for each SHIV, and we performed a pool competition study in rhesus monkeys to define the optimal variant for each SHIV prior to generating large-scale challenge stocks. We identified SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH as the optimal variants. SHIV-SF162p3S could not be improved, as it already contained the optimal Env375 residue. SHIV-AE16W exhibited a similar replicative capacity to the parental SHIV-AE16 stock. In contrast, SHIV-325cH demonstrated a 2.6-log higher peak and 1.6-log higher setpoint viral loads than the parental SHIV-325c stock. These data demonstrate the diversity of potential outcomes following Env375 modification in SHIVs. Moreover, the clade C SHIV-325cH challenge stock may prove useful for evaluating prophylactic or therapeutic interventions against clade C HIV-1.

IMPORTANCE We sought to enhance the infectivity of three SHIV stocks by optimization of a key residue in human immunodeficiency virus type 1 (HIV-1) Env (Env375). We developed the following three new simian-human immunodeficiency virus (SHIV) stocks: SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH. SHIV-SF162p3S could not be optimized, SHIV-AE16W proved comparable to the parental virus, and SHIV-325cH demonstrated markedly enhanced replicative capacity compared with the parental virus.

Existing research shows that distrust of the police is widespread and consequential for public safety. However, there is a shortage of interventions that demonstrably reduce negative police interactions with the communities they serve. A training program in Chicago attempted to encourage 8,480 officers to adopt procedural justice policing strategies. These...

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme...

Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such “early activation” genes silent have been a mystery. In the course...

Background:

Thyroid disorders are among the most commonly treated conditions by the United States health care system. The number of patients reporting thyroid hormone use has increased in recent years, but it is unknown if there have been differential increases in the number of treated individuals within different demographic groups. Previous research has also not evaluated how expenditures for different thyroid hormone medications have changed in recent years.

Ewa Komar, Dina K. N. Dechmann, Nicolas J. Fasel, Marcin Zegarek, and Ireneusz Ruczynski

Balancing energy budgets can be challenging, especially in periods of food shortage, adverse weather conditions and increased energy demand due to reproduction. Bats have particularly high energy demands compared to other mammals and regularly use torpor to save energy. However, while torpor limits energy expenditure, it can also downregulate important processes, such as sperm production. This constraint could result in a trade-off between energy saving and future reproductive capacity. We mimicked harsh conditions by restricting food and tested the effect on changes in body mass, torpor use and seasonal sexual maturation in male parti-coloured bats (Vespertilio murinus). Food-restricted individuals managed to maintain their initial body mass, while in well-fed males, mass increased. Interestingly, despite large differences in food availability, there were only small differences in torpor patterns. However, well-fed males reached sexual maturity up to half a month earlier. Our results thus reveal a complex trade-off in resource allocation; independent of resource availability, males maintain a similar thermoregulation strategy and favour fast sexual maturation, but limited resources and low body mass moderate this latter process.

Background

Mutations in CTNS—a gene encoding the cystine transporter cystinosin—cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments.

BACKGROUND:The N95 filtering facepiece respirator (FFR) is the most popular individual protective device to reduce exposure to particulate matter. However, concerns have been raised with regard to its use because it can increase respiratory resistance and dead space. Therefore, this study assessed the safety of N95 use in patients with COPD and air-flow limitation.METHODS:This prospective study was performed at a tertiary hospital and enrolled 97 subjects with COPD. The subjects were monitored for symptoms and physiologic variables during a 10-min rest period and 6-min walking test while wearing an N95.RESULTS:Of the 97 subjects, 7 with COPD did not wear the N95 for the entire test duration. This mask-failure group showed higher British modified Medical Research Council dyspnea scale scores and lower FEV1 percent of predicted values than did the successful mask use group. A modified Medical Research Council dyspnea scale score ≥ 3 (odds ratio 167, 95% CI 8.4 to >999.9; P = .008) or a FEV1 < 30% predicted (odds ratio 163, 95% CI 7.4 to >999.9; P = .001) was associated with a risk of failure to wear the N95. Breathing frequency, blood oxygen saturation, and exhaled carbon dioxide levels also showed significant differences before and after N95 use.CONCLUSIONS:This study demonstrated that subjects with COPD who had modified Medical Research Council dyspnea scale scores ≥ 3 or FEV1 < 30% predicted wear N95s only with care.

BACKGROUND:Assisted coughing via mechanical in-exsufflation (MI-E) is a first-line treatment for secretion management in patients with amyotrophic lateral sclerosis (ALS) with unassisted CPF < 4.25 L/s. Some devices enable oscillations to be added to MI-E (MI-E+O). We sought to determine whether adding oscillations to MI-E enables a reduction in the use of invasive secretion management procedures (ie, bronchoscopy or tracheostomy) in subjects with ALS.METHODS:We conducted a 12-month, prospective, randomized follow-up study of subjects with ALS for whom assisted coughing techniques were indicated. One group was treated with oscillations in addition to MI-E (MI-E+O), and the other group was treated with conventional MI-E.RESULTS:29 subjects were included in the MI-E group and 27 subjects were included in the MI-E+O group. Five subjects (8.9%) required invasive techniques for secretion management (3 in the MI-E group and 2 in the MI-E+O group, P = .70). Treatment with MI-E+O did not alter the risk of invasive procedures (odds ratio 0.69, 95% CI 0.10–4.50, P = .70). The mean number of respiratory infections was 0.58 ± 0.16 in the MI-E group and 0.025 ± 0.08 in the MI-E+O group (P = .10). Survival was 8.96 ± 0.18 months in the MI-E group and 7.70 ± 0.70 months in the MI-E+O group (P = .10).CONCLUSION:Adding oscillations to MI-E did not enable a reduction in the need to perform invasive procedures for secretion management in subjects with ALS.

Drug-induced liver injury (DILI) is a global medical problem. The risk of DILI is often related to expression and activities of drug-metabolizing enzymes, especially cytochrome P450s (P450s). However, changes on expression and activities of P450s after DILI have not been determined. The aim of this study is to fill this knowledge gap. Acetaminophen (APAP) was used as a model drug to induce DILI in C57BL/6J mice at different ages of days 10 (infant), 22 (child), and 60 (adult). DILI was assessed by levels of alanine aminotransferase and aspartate aminotransferase in plasma with a confirmation by H&E staining on liver tissue sections. The expression of selected P450s at mRNA and protein levels was measured by real-time polymerase chain reaction and liquid chromatography–tandem mass spectrometry, respectively. The activities of these P450s were determined by the formation of metabolites from probe drugs for each P450 using ultraperformance liquid chromatography–quadrupole time of flight mass spectrometry. DILI was induced at mild to severe levels in a dose-dependent manner in 200, 300, and 400 mg/kg APAP-treated groups at child and adult ages, but not at the infant age. Significantly decreased expression at mRNA and protein levels as well as enzymatic activities of CYP2E1, 3A11, 1A2, and 2C29 were found at child and adult ages. Adult male mice were more susceptible to APAP-induced liver injury than female mice with more decreased expression of P450s. These results suggest that altered levels of P450s in livers severely injured by drugs may affect the therapeutic efficacy of drugs, which are metabolized by P450s, more particularly for males.

The DOCK-D (dedicator of cytokinesis D) family proteins are atypical guanine nucleotide exchange factors that regulate Rho GTPase activity. The family consists of Zizimin1 (DOCK9), Zizimin2 (DOCK11), and Zizimin3 (DOCK10). Functions of the DOCK-D family proteins are presently not well-explored, and the role of the DOCK-D family in neuroinflammation is unknown. In this study, we generated three mouse lines in which DOCK9 (DOCK9−/−), DOCK10 (DOCK10−/−), or DOCK11 (DOCK11−/−) had been deleted and examined the phenotypic effects of these gene deletions in MOG35–55 peptide-induced experimental autoimmune encephalomyelitis, an animal model of the neuroinflammatory disorder multiple sclerosis. We found that all the gene knockout lines were healthy and viable. The only phenotype observed under normal conditions was a slightly smaller proportion of B cells in splenocytes in DOCK10−/− mice than in the other mouse lines. We also found that the migration ability of macrophages is impaired in DOCK10−/− and DOCK11−/− mice and that the severity of experimental autoimmune encephalomyelitis was ameliorated only in DOCK10−/− mice. No apparent phenotype was observed for DOCK9−/− mice. Further investigations indicated that lipopolysaccharide stimulation up-regulates DOCK10 expression in microglia and that microglial migration is decreased in DOCK10−/− mice. Up-regulation of C–C motif chemokine ligand 2 (CCL2) expression induced by activation of Toll-like receptor 4 or 9 signaling was reduced in DOCK10−/− astrocytes compared with WT astrocytes. Taken together, our findings suggest that DOCK10 plays a role in innate immunity and neuroinflammation and might represent a potential therapeutic target for managing multiple sclerosis.

Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce α-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble α-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled α-synuclein in these different samples. We conclude that α-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.

Site-specific recombinases, such as Cre, are a widely used tool for genetic lineage tracing in the fields of developmental biology, neural science, stem cell biology, and regenerative medicine. However, nonspecific cell labeling by some genetic Cre tools remains a technical limitation of this recombination system, which has resulted in data misinterpretation and led to many controversies in the scientific community. In the past decade, to enhance the specificity and precision of genetic targeting, researchers have used two or more orthogonal recombinases simultaneously for labeling cell lineages. Here, we review the history of cell-tracing strategies and then elaborate on the working principle and application of a recently developed dual genetic lineage-tracing approach for cell fate studies. We place an emphasis on discussing the technical strengths and caveats of different methods, with the goal to develop more specific and efficient tracing technologies for cell fate mapping. Our review also provides several examples for how to use different types of DNA recombinase–mediated lineage-tracing strategies to improve the resolution of the cell fate mapping in order to probe and explore cell fate–related biological phenomena in the life sciences.

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene–Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome–manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain–containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from the manchette to sculpt sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3 knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examination revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicate that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope, required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues regarding the underlying pathology of the globozoospermia-like phenotype.

Current pneumococcal vaccines cover the 10 to 23 most common serotypes of the 92 presently described. However, with the increased usage of pneumococcal-serotype-based vaccines, the risk of serotype replacement and an increase in disease caused by nonvaccine serotypes remains. Serotype surveillance of pneumococcal infections relies heavily on culture techniques, which are known to be insensitive, particularly in cases of noninvasive disease. Pneumococcal-serotype-specific urine assays offer an alternative method of serotyping for both invasive and noninvasive disease. However, the assays described previously cover mainly conjugate vaccine serotypes, give little information about circulating nonvaccine serotypes, and are currently available only in one or two specialist laboratories. Our laboratory has developed a Luminex-based extended-range antigen capture assay to detect pneumococcal-serotype-specific antigens in urine samples. The assay targets 24 distinct serotypes/serogroups plus the cell wall polysaccharide (CWP) and some cross-reactive serotypes. We report that the assay is capable of detecting all the targeted serotypes and the CWP at 0.1 ng/ml, while some serotypes are detected at concentrations as low as 0.3 pg/ml. The analytical serotype specificity was determined to be 98.4% using a panel of polysaccharide-negative urine specimens spiked with nonpneumococcal bacterial antigens. We also report clinical sensitivities of 96.2% and specificities of 89.9% established using a panel of urine specimens from patients diagnosed with community-acquired pneumonia or pneumococcal disease. This assay can be extended for testing other clinical samples and has the potential to greatly improve serotype-specific surveillance in the many cases of pneumococcal disease in which a culture is never obtained.

The kinetics, longevity, and breadth of antibodies to influenza virus neuraminidase (NA) in archival, sequential serum/plasma samples from influenza A virus (IAV) H5N1 infection survivors and from patients infected with the 2009 pandemic IAV (H1N1) virus were determined using an enzyme-linked lectin-based assay. The reverse-genetics-derived H4N1 viruses harboring a hemagglutinin (HA) segment from A/duck/Shan Tou/461/2000 (H4N9) and an NA segment derived from either IAV H5N1 clade 1, IAV H5N1 clade 2.3.4, the 2009 pandemic IAV (H1N1) (H1N1pdm), or A/Puerto Rico/8/1934 (H1N1) virus were used as the test antigens. These serum/plasma samples were also investigated by microneutralization (MN) and/or hemagglutination inhibition (HI) assays. Neuraminidase-inhibiting (NI) antibodies against N1 NA of both homologous and heterologous viruses were observed in H5N1 survivors and H1N1pdm patients. H5N1 survivors who were never exposed to H1N1pdm virus developed NI antibodies to H1N1pdm NA. Seroconversion of NI antibodies was observed in 65% of the H1N1pdm patients at day 7 after disease onset, but an increase in titer was not observed in serum samples obtained late in infection. On the other hand, an increase in seroconversion rate with the HI assay was observed in the follow-up series of sera obtained on days 7, 14, 28, and 90 after infection. The study also showed that NI antibodies are broadly reactive, while MN and HI antibodies are more strain specific.

Accurate detection of influenza A virus (IAV) is crucial for patient management, infection control, and epidemiological surveillance. The World Health Organization and the Centers for Disease Control and Prevention have recommended using the M gene as the diagnostic gene target for reverse-transcription-PCR (RT-PCR). However, M gene RT-PCR has reduced sensitivity for recent IAV due to novel gene mutations. Here, we sought to identify novel diagnostic targets for the molecular detection of IAV using long-read third-generation sequencing. Direct nanopore sequencing from 18 nasopharyngeal specimens and one saliva specimen showed that the 5' and 3' ends of the PB2 gene and the entire NS gene were highly abundant. Primers selected for PB2 and NS genes were well matched with seasonal or avian IAV gene sequences. Our novel PB2 and NS gene real-time RT-PCR assays showed limits of detection similar to or lower than that of M gene RT-PCR and achieved 100% sensitivity and specificity in the detection of A(H1N1), A(H3N2), and A(H7N9) in nasopharyngeal and saliva specimens. For 10 patients with IAV detected by M gene RT-PCR conversion in sequentially collected specimens, NS and/or PB2 gene RT-PCR was positive in 2 (20%) of the initial specimens that were missed by M gene RT-PCR. In conclusion, we have shown that PB2 or NS gene RT-PCRs are suitable alternatives to the recommended M gene RT-PCR for diagnosis of IAV. Long-read nanopore sequencing facilitates the identification of novel diagnostic targets.

Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. The capability of PCR followed by high-resolution melt (HRM) curve analysis for the detection of community-associated and livestock-associated MRSA strains and the identification of staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans, and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A nonsubjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings, and result data were reanalyzed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies.

The QIAstat-Dx Respiratory Panel (QIAstat-Dx RP) is a multiplex in vitro diagnostic test for the qualitative detection of 20 pathogens directly from nasopharyngeal swab (NPS) specimens. The assay is performed using a simple sample-to-answer platform with results available in approximately 69 min. The pathogens identified are adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, human metapneumovirus A and B, influenza A, influenza A H1, influenza A H3, influenza A H1N1/2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus/enterovirus, respiratory syncytial virus A and B, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae. This multicenter evaluation provides data obtained from 1,994 prospectively collected and 310 retrospectively collected (archived) NPS specimens with performance compared to that of the BioFire FilmArray Respiratory Panel, version 1.7. The overall percent agreement between QIAstat-Dx RP and the comparator testing was 99.5%. In the prospective cohort, the QIAstat-Dx RP demonstrated a positive percent agreement of 94.0% or greater for the detection of all but four analytes: coronaviruses 229E, NL63, and OC43 and rhinovirus/enterovirus. The test also demonstrated a negative percent agreement of ≥97.9% for all analytes. The QIAstat-Dx RP is a robust and accurate assay for rapid, comprehensive testing for respiratory pathogens.

Serological testing for nasopharyngeal carcinoma (NPC) has recently been reinvigorated by the implementation of novel Epstein-Barr virus (EBV)-specific IgA and IgG antibodies from a proteome array. Although proteome arrays are well suited for comprehensive antigen selection, they are not applicable for large-scale studies. We adapted a 13-marker EBV antigen signature for NPC risk identified by proteome arrays to multiplex serology to establish an assay for large-scale studies. Taiwanese NPC cases (n = 175) and matched controls (n = 175) were used for assay validation. Spearman’s correlation was calculated, and the diagnostic value of all multiplex markers was assessed independently using the area under the receiver operating characteristic curve (AUC). Two refined signatures were identified using stepwise logistic regression and internally validated with 10-fold cross validation. Array and multiplex serology showed strong correlation for each individual EBV marker, as well as for a 13-marker combined model on continuous data. Two refined signatures with either four (LF2 and BGLF2 IgG, LF2 and BMRF1 IgA) or two (LF2 and BGLF2 IgG) antibodies on dichotomous data were identified as the most parsimonious set of serological markers able to distinguish NPC cases from controls with AUCs of 0.992 (95% confidence interval [CI], 0.983 to 1.000) and 0.984 (95% CI, 0.971 to 0.997), respectively. Neither differed significantly from the 13-marker model (AUC, 0.992; 95% CI, 0.982 to 1.000). All models were internally validated. Multiplex serology successfully validated the original EBV proteome microarray data. Two refined signatures of four and two antibodies were capable of detecting NPC with 99.2% and 98.4% accuracy.

Genome-wide association studies have implicated thousands of noncoding variants across common human phenotypes. However, they cannot directly inform the cellular context in which disease-associated variants act. Here, we use open chromatin profiles from discrete mouse cell populations to address this challenge. We applied stratified linkage disequilibrium score regression and evaluated heritability enrichment in 64 genome-wide association studies, emphasizing schizophrenia. We provide evidence that mouse-derived human open chromatin profiles can serve as powerful proxies for difficult to obtain human cell populations, facilitating the illumination of common disease heritability enrichment across an array of human phenotypes. We demonstrate that signatures from discrete subpopulations of cortical excitatory and inhibitory neurons are significantly enriched for schizophrenia heritability with maximal enrichment in cortical layer V excitatory neurons. We also show that differences between schizophrenia and bipolar disorder are concentrated in excitatory neurons in cortical layers II-III, IV, and V, as well as the dentate gyrus. Finally, we leverage these data to fine-map variants in 177 schizophrenia loci nominating variants in 104/177. We integrate these data with transcription factor binding site, chromatin interaction, and validated enhancer data, placing variants in the cellular context where they may modulate risk.

We hypothesized that, in mice, histamine via the histamine receptor subtype 4 (H₄R) on colon epithelial cells affects epithelial barrier integrity, perturbing physiologic function of the colonic mucosa and thus aggravating the severity of colitis. To test this hypothesis, bone marrow–chimeric mice were generated from H₄R knockout (H₄R^–/–) and wild-type (WT) BALB/cJ mice and subjected to the dextrane sodium sulfate (DSS)-induced acute colitis model. Clinical symptoms and pathohistological derangements were scored. Additionally, total RNA was extracted from either mouse whole-colon homogenates or primary cell preparations enriched for epithelial cells, and gene expression was analyzed by real-time quantitative polymerase chain reaction. The impact of the H₄R on epithelial barrier function was assessed by measurement of transepithelial electrical resistence of organoid-derived two-dimensional monolayers from H₄R^–/– and WT mice using chopstick electrodes. Bone marrow–chimeric mice with genetic depletion of the H₄R in nonhematopoietic cells exhibited less severe DSS-induced acute colitis symptoms compared with WT mice, indicating a functional proinflammatory expression of H₄R in nonimmune cells of the colon. Analysis of H₄R expression revealed the presence of H₄R mRNA in colon epithelial cells. This expression could be confirmed and complemented by functional analyses in organoid-derived epithelial cell monolayers. Thus, we conclude that the H₄R is functionally expressed in mouse colon epithelial cells, potentially modulating mucosal barrier integrity and intestinal inflammatory reactions, as was demonstrated in the DSS-induced colitis model, in which presence of the H₄R on nonhematopoietic cells aggravated the inflammatory phenotype.

Tobacco use is a persistent public health issue. It kills up to half its users and is the cause of nearly 90% of all lung cancers. The main psychoactive component of tobacco is nicotine, primarily responsible for its abuse-related effects. Accordingly, most pharmacotherapies for smoking cessation target nicotinic acetylcholine receptors (nAChRs), nicotine’s major site of action in the brain. The goal of the current review is twofold: first, to provide a brief overview of the most commonly used behavioral procedures for evaluating smoking cessation pharmacotherapies and an introduction to pharmacokinetic and pharmacodynamic properties of nicotine important for consideration in the development of new pharmacotherapies; and second, to discuss current and potential future pharmacological interventions aimed at decreasing tobacco use. Attention will focus on the potential for allosteric modulators of nAChRs to offer an improvement over currently approved pharmacotherapies. Additionally, given increasing public concern for the potential health consequences of using electronic nicotine delivery systems, which allow users to inhale aerosolized solutions as an alternative to smoking tobacco, an effort will be made throughout this review to address the implications of this relatively new form of nicotine delivery, specifically as it relates to smoking cessation.